The long-term goal of the project is to investigate the role of p53 in osteoblast differentiation, a process central to bone formation. In our studies with osteosarcomas, we have shown that functional loss of p53 predominates, and replacing the wild type p53 gene into these cells not only results in suppression of growth, but in the induction of osteoblast differentiation. We propose that p53 provides the right internal environment for differentiation by factoring the input from several sources, due to its involvement in cellular pathways like apoptosis and cell cycle arrest. The proposed experiments are therefore designed to understand: a) how p53 is regulated during osteoblast differentiation. b) The type of regulation that allows the maintenance of p53 expression. c) The type of p53 mediated regulation that allows for expression of bone specific targets. The focus of the research will be: a). To identify p53 regulation during osteoblast differentiation. We hypothesize that p53 regulation during differentiation is transcriptional unlike DNA damage and stress where the protein is stabilized. We will confirm its mechanism of activation during in vitro bone differentiation, define post-translational changes that occur to p53 during differentiation and also demonstrate its regulation by bone specific agents. b).We hypothesize that p53's role in osteoblast differentiation is mediated by its ability to associate with other regulatory proteins that have an effect on bone differentiation. We hypothesize that this is the default pathway which when perturbed activates either cell cycle arrest or apoptosis. We will demonstrate this by studying the role of p300 in osteoblast differentiation. We will also determine association of p53 with other regulatory proteins during differentiation and if competition for p300 by p53 and other proteins determine the balance between growth and differentiation. c).Validate targets of p53 inducible genes. While a number of genes identified as p53 targets have important roles in bone, it is not known if they function in a p53 regulated manner during differentiation. We will study targets that have been identified to have p53 response elements to determine if they undergo p53 dependent regulation during in vitro bone differentiation. We anticipate that the proposed studies will generate valuable new data, shed more light on the physiological role of the p53 protein, and advance our knowledge of the mechanisms responsible for osteoblast cell differentiation. A better understanding of this basic aspect of cell biology could also potentially lead to additional benefits in diseases involving bone and cancer. The information generated would allow us to better understand p53's loss in primary osteosarcomas and its presence in advanced prostate cancers with propensity to form osteoblastic lesions in bone, and its mutations in conditions of bone destruction (rheumatoid arthritis). Investigation into the role of p53 in osteoblast differentiation will provide a better understanding of this aspect of basic cell biology and could also potentially lead to additional benefits in treating diseases involving bone and cancer. [unreadable] [unreadable] [unreadable]